Techniques for studying the in vivo conformation of chromosomes can reveal a wealth of information regarding mechanisms of gene regulation. For example, chromosome conformation studies can reveal long-range regulatory control of genes by distal DNA sequence elements. One widely used methodology for determining the in vivo conformation of chromosomes is called chromosome conformation capture, or 3C. Other techniques extend 3C to provide further information or enable higher throughput analysis. Such techniques include, for example, 4C, 5C, 6C, Hi-C, ChIP-loop, and ChIA-PET.
Generally, chromosome conformation capture techniques involve a cross-linking step to fix the three-dimensional organization of chromatin, an endonuclease digestion step, a ligation step, and a reverse crosslinking step to generate a library of nucleic acid molecules. This library contains nucleic acid molecules in which sequence elements that were in proximity due to three-dimensional chromatin structure prior to the cross-linking event become proximal at the primary structural level after the reverse cross-linking event. Various amplification, sequencing, or other nucleic acid detection methods can then be utilized to identify the sequence elements that were proximal in three-dimensional space.
The ligation step can require extensive optimization to provide a suitable library of nucleic acid molecules. For example, intramolecular ligation events, in which cross-linked elements are ligated so that they exist on the same nucleic acid molecule, are generally favored. In contrast, it is generally desirable to avoid or minimize intermolecular ligation events, in which sequence elements that were not cross-linked become ligated. As another example, it can be important to ensure that no ligation occurs after reverse cross-linking. As yet another example, insufficient ligation can result in a library of molecules that provides little information.